Enzymes are biologically active proteins that catalyze certain reactions. Enzymes are useful in many industrial and research applications, such as fermentation, food processing, and medical research.
Since enzymes are commonly water soluble as well as being generally unstable, they are subject to deactivation and are difficult to remove for re-use from aqueous solutions in which they are utilized. These difficulties have led to an increased cost in the use of enzymes in commercial scale operations due to the necessity for their frequent replacement. In order to reduce the high cost of enzyme replacement, various methods to immobilize enzymes or enzyme-producing cells prior to use have been devised. This immobilization permits reuse whereas otherwise the enzyme might undergo deactivation or be lost in the reaction medium. The immobilized conjugate may be employed directly in a substrate solution or in a reactor system such as a packed column, depending on the nature of the substrate which is being biocatically reacted.
Several general methods as well as many modifications of the methods have been described in the literature by which immobilization can be effected. For example, materials useful for immobilization of solubilized enzymes or cells containing enzymes are disclosed in U.K. Patent No. 1,444,539. Preferably, the enzymes or cells are treated with a water miscible solvent, such as acetone, dried and then treated with polyethylenimine and glutaraldehyde to make shaped bodies of a water insoluble structure.
Also, in U.S. Pat. No. 4,078,970 there is disclosed the immobilization of glucose isomerase, preferably obtained from cells belonging to the genus, Streptomyces. Release of the intracellular glucose isomerase from the microbial cells is conducted by any of various known processes for disruption of the cells, such as shearing in a blender, autolysis, lysozyme treatment, ultra-sound treatment, pressurized treatment, or detergent treatment. Cell debris is then removed (typically by centrifuging with decantation of the enzyme-containing solution), and the now released glucose isomerase is adsorbed on a macroporous anion exchange resin. The resin is prepared by a known process. For instance, a crosslinkable monomer and a monovinyl monomer are copolymerized in the presence of a material which is removable by a solvent and does not take part in the reaction, such as polystyrene. After polymerization, the resin is treated with solvent to dissolve the polystyrene therefrom. Then, an anion exchange group, such as trimethyl ammonium or dimethyl ammonium, is introduced. For clarity, it is mentioned here that U.S. Pat. No. 4,078,970 uses the term "immobilized" glucose isomerase interchangeably with the term "insolubilized" glucose isomerase to refer to when the glucose isomerase is on the resin support. This should not be confused with the generally accepted terminology in the art of enzyme chemistry to use the term "solubilized" to refer to when an enzyme has been released from the cell and is in solution. Thus, "solubilized" refers to when an extracellular enzyme has been secreted through the cell wall to outside the cell or to when an intracellular enzyme has been brought outside the cell by disruption means such as detergent treatment, sonication, shearing with a blender, and the like.
Similarly, U.S. Pat. Nos. 3,788,945 and 3,960,663 disclose released glucose isomerase (free of cell debris) adsorbed on a porous anion exchange resin such as Amberlite.RTM. IRA 938, Amberlite.RTM. IRA 900 and Amberlite.RTM. IRA 904.
Additionally, in Japanese Kokai Patent No. Sho 51 [1976]-128,474 a glucose isomerase immobilization method which involves the blending of chitosan and glucose isomerase producing bacteria, with subsequent treatment with a polyaldehyde, is disclosed. Immobilization of cells of glucose isomerase producing Streptomyces olivaceus by contacting the cells with glutaraldehyde and a cationic polymer obtained from polymerizing epihalohydrin and an alkylene polyamine to obtain a hard cell aggregate is disclosed in U.S. Pat. No. 4,251,632. On the other hand, immobilization of glucose isomerase producing cells by contacting the cells with glutaraldehyde, a copolymer of epihalohydrin and polyamine, and cellulose or natural gum as binder, and then spheronizing the preparation to obtain a cell aggregate of increased hardness is disclosed in U.S. Pat. No. 4,543,332.
Lastly, a process for depositing and immobilizing various enzymes by causing an aqueous dispersion of the enzyme to flow through an inert, inorganic, porous, sorptive, liquid permeable, metal oxide, supporting matrix is disclosed in U.S. Pat. No. 4,001,085. In the preferred embodiment, the matrix is ceramic and formed by sintering alumina. The porous matrix is enzyme permeable, and the patent discloses a process to deposit the enzyme on the matrix by applying at least 10 psig (1.736 kg/cm.sup.2) pressure to cause a dispersion of the enzyme to flow through the matrix. Although this patent discloses many enzymes, it does not mention or suggest immobilization of enzyme-containing cells.
No where in the prior art is there disclosed an active immobilized cell conjugate comprising a strain of enzyme-containing microorganism cells on a macroporous resin, wherein said resin comprises a less expensive, uncharged (non-ionic) resin support. The catalytic activity is not altered as it is when positive or negative charges are present.